A COMPOSITION USEFUL AS ADDITIVE TO A CELL CULTURE MEDIUM, COMPRISING COLOSTRUM AND SERUM

Abstract: The invention concerns a composition useful as additive to culture media for the cultivation of adherent cells, said composition comprising a colostrum fraction and a serum component in an amount sufficient to enhance the proliferation of cells after the addition of a suitable amount of said composition to a culture medium. The invention further concerns culture media comprising a colostrum fraction and serum as well as the use of such culture media for the cultivation of adherent cells.

Description: A composition useful as additive to a cell culture medium, comprising colostrum and serum FIELD OF THE INVENTION This invention relates to a novel composition useful as additive to culture media for the cultivation of adherent cells. The invention concerns further novel culture media and their use in the cultivation of such cells. BACKGROUND OF THE INVENTION Cell culture media for large-scale production of various biochemicals such as monoclonal antibodies, growth factors, hormones, vaccines and substances to be used in therapy must fulfil several important criteria: 1. Substance production should be continuous and reproducible. 2. Process costs should be as low as possible. 3. Risk of contamination by infectious agents and endotoxins should be eliminated. In the cultivation of animal cells, a basal medium is traditionally supplemented with mammal blood serum. Such serum contains several partly unknown cell growth promoting agents, such as growth factors, vitamins, trace elements, hormones, binding proteins and attachment factors. A serum suitable for most purposes is fetal bovine serum (FBS). The price of this serum has increased greatly in recent years because of the increasing demand and the limited availability of sufficiently pure sera. The complexity, limited availability and high price of FBS and serious drawbacks in using FBS especially in large scale bioreactor has led to efforts aiming at eliminating FBS as a nutritional supplement. In some cases, FBS can be replaced with newborn calf serum, calf serum or even adult bovine serum. FBS is, however, most effective. This is probably due to its high growth factor content. Other important proteins in serum are attachment factors. These include, for example, fibronectin. Most adherent cell types require additional attachment factors in cell culture medium although some cells can also synthezise them by themselves. If the objective of growing animal cells is the production, purification and characterization of secreted compounds such as monoclonal antibodies, the use of serum-free medium containing reduced amounts of protein and immunoglobulins makes downstream processing more simple and can greatly improve end-product purification and recovery. In a paper by R Pakkanen et al. , Appl Microbiol Biotechnol (1992) 37: 451 - 456 has been described a bovine colostrum fraction useful as serum substitute for the cultivation of mouse hybridomas for production of monoclonal antibodies. Fractions of bovine colostrum were prepared and their ability to support the growth of mouse-mouse hybridomas in culture was tested. Whey was prepared from defatted colostrum by removal of casein using acid precipitation. An ultrafiltrate was obtained from cleared whey by filtration through membranes with a nominal molecular weight cut-off of 100000 Da. The colostrum ultrafiltrate (colostrum fraction) so obtained contained 1.16 g/1 protein, 0.24 g/1 im unoglobulin G (IgG) and less than 0.24 EU (endotoxin unit)/ml endotoxins. The effect of defatted colostrum, whey and ultrafiltrate as serum substitutes was studied by cultivation of hybridoma cells in minimal essential medium containing different concentrations of supplements. Under optimal conditions in ultrafiltrate-supplemented medium, the maximal cell concentration was 35 - 40 % of that obtained using 10 % fetal bovine serum, and antibody production per cell was equal to that achieved using fetal bovine serum. The results of this study showed that the bovine colostrum ultrafiltrate provides a very attractive alternative to fetal bovine serum for production of monoclonal antibodies. An important group of cells used for the production of biochemicals are classified as adherent cells. Animal cells can be divided into two classes according to the manner in which they grow: suspension cells, which grow in suspended form, and adherent cells, which grow attached to a surface. Hybrido a cells are typically suspension cells while most of the other cells could be classified as adherent cells. Typical examples of adherent cells are e.g. Vero (kidney cells of the African Green Monkey), CHO-K1 (ovary cell line of the Chinese hamster) and 3T3-fibroblast. The Vero and CHO-K1-cells are widely used in the biotechnical industry. Vero cells are used in the production of viruses and viral proteins and CHO-K1- and CHO-cells are often used as host cells in large scale production of recombinant proteins. Bovine colostrum resembles FBS in the respect that it contains growth factors. The growth factor content of colostrum is high. As reported in the above study by R Pakkanen et al. , a colostrum ultrafiltrate could be used to replace FBS in hybridoma cell cultures. On the other hand, colostrum does not contain enough attachment factors for many adherent cells. Such attachment factors can be found in FBS as well as in bovine serum (BS). The effect of the colostrum ultrafiltrate fraction on the growth of adherent cells has also been tested. Vero and CHO-K1 cells were grown in a basal medium containing varying amounts of colostrum ultrfiltrate according to the method described in the paper by R Pakkanen et al. A minor effect could be observed at colostrum ultrafiltrate concentrations ranging from 10 to 15 %, but the effect was very small compared to that caused to the growth of the hybridoma cells. It seems therefore clear that the replacement of fetal bovine serum with a colostrum fraction (colostrum ultrafiltrate) as additive to the culture medium does not lead to satisfactory results in the cultivation of adherent cells. As mentioned above, bovine serum contains the important attachment factor fibronectin. According to US 4,440,860, fibronectin added to colostrum possesses an advantageous effect on the proliferation of adherent cells. However, as will be seen below, the addition of fibronectin to a colostrum fraction according to this invention did not affect the growth of adherent cells. OBJECT OF THE INVENTION It is therefore an object of the invention to provide a composition which mixed with the basal medium gives a highly efficient culture medium for the cultivation of adherent cells. Another object of the invention is to provide an efficient culture medium for the cultivation of such cells. Yet another object of the invention is to provide a method for the growth of adherent cells, by utilizing the culture medium of the invention. BRIEF DESCRIPTION OF THE DRAWINGS The Figures IA to 6 describes the number of viable cells versus time during continuous cultivation on a culture medium comprising different attachment factor additive compositions and different amounts thereof. Unless otherwise specified in the presentation of the particular Figures, in the experiments on which the Figures are based the the basal media used were DMEM for Vero cells and DMEM- F12 (1:1) for CHO-K1 cells supplemented with glutamine (4 mM), penicillin (100 units/ml) and streptomycin (100 μg/ml). Unless otherwise specified in the presentation of the particular Figures, in the experiments human transferrin (hTF) was added to the culture medium to give a concentration of 5 mg/1. All the percentages below are vol- %. For further details reference is made to the experiments. Figure IA shows the number of viable CHO-Kl-cells versus time in a culture medium supplemented with 10 % FBS (•) and 13 % bovine colostrum ultrafiltrate (in the following abbreviated UF) (0). Figure IB shows the number of viable Vero-cells versus time in a culture medium supplemented with 10 % FBS (•) and 13 % colostrum ultrafiltrate (in the following abbreviated UF) (0). Figure 2A shows the number of viable CHO-Kl-cells versus time in a culture medium supplemented with 2 % BS (bovine serum) + 13 % UF (X) 1 % BS + 13 % UF (_i) 0.6 % BS + 13 % UF (■) 0.33 % BS + 13 % UF (▼) 0.1 % BS + 13 % UF (•) Figure 2B shows the number of viable Vero-cells versus time in a culture medium supplemented with 2 % BS + 13 % UF (X) 1 % BS + 13 % UF (A) 0.6 % BS + 13 % UF (p) 0.33 % BS + 13 % UF ( ▼ ) 0.1 % BS + 13 % UF (t) Figure 3A shows the number of viable CHO-Kl-cells versus time in a culture medium supplemented with 1 % BS + 13 % UF (0) 1 % BS + 10 % UF (v) 1 % BS + 6.67 % UF (π) 1 % BS + 3.3 % UF (A) Figure 3B shows the number of viable Vero-cells versus time in a culture medium supplemented with 1 % BS + 13 % UF (•) 1 % BS + 10 % UF ( ) 1 % BS + 6.7 % UF (■) 1 % BS + 3.3 % UF (_4) Figure 4A shows the number of viable CHO-Kl-cells versus time in a culture medium supplemented with 0.5 % BS, no hTF added (•) 1.0 % BS, " (▼) 2.0 % BS, " (■) 1.0 % BS + 6.7 % UF, 5 mg/1 hTF (0) 0.33 % BS + 6.7 % UF, 5 mg/1 hTF (v) 0.60 % BS + 6.7 % UF, 5 mg/1 hTF (D) 1.0 % BS + 10 % UF, 5 mg/1 hTF ( ) Figure 4B shows the number of viable Vero-cells versus time in a culture medium supplemented with 0.5 % BS, no hTF added (t) 1.0 % BS, " ( ▼ ) 2.0 % BS, " (m) 0.66 % BS + 10 % UF, 5 mg/1 hTF (0) 0.33 % BS + 6.7 % UF, 5 mg/1 hTF (v) 0.60 % BS + 6.7 % UF, 5 mg/1 hTF (D) 1.0 % BS + 10 % UF, 5 mg/1 hTF (_Δ) Figure 5A shows the number of viable CHO-Kl-cells versus time in a culture medium supplemented with 0.5 % FBS, no hTF added (•) 1.0 % FBS, " (ψ) 2.0 % FBS, no hTH added ( B ) 10 % FBS, no hTF added (_4) 1.0 % BS + 6.7 % UF, 5 mg/1 hTF (0) 0.33 % BS + 6.7 % UF, 5 mg/1 hTF (V) 0.60 % BS + 6.7 % UF, 5 mg/1 hTF (D) 1.0 % BS + 10 % UF, 5 mg/1 hTF ( .) no supplement of BS, UF or hTF added (X) Figure 5B shows the number of viable Vero-cells versus time in a culture medium supplemented with 0.5 % FBS, no hTF added (•) 1.0 % FBS, " (ψ) 2.0 % FBS, " (■) 10 % FBS, " (A) 0.66 % BS + 10 % UF, 5 mg/1 hTF (0) 0.33 % BS + 6.7 % UF, 5 mg/1 hTF (V) 0.60 % BS + 6.7 % UF, 5 mg/1 hTF (D) 1.0 % BS + 10 % UF, 5 mg/1 hTF (__.) no supplement of BS, UF or hTF added (X) Figure 6 shows the number of viable CHO-Kl-cells versus time in a culture medium supplemented with 10 % FBS (•) 10 % UF (0) 10 % UF + 65 μg/ml of fibronectin (D) DETAILED DESCRIPTION OF THE INVENTION It has now surprisingly been found that a composition comprising a colostrum fraction, e.g. the colostrum ultrafiltrate as described in the experiments, and small amounts of serum efficiently enhances the growth of adherent cells when added to the culture medium for said cells. The results obtained clearly exhibit a synergistic effect obtained by combining the two components, i.e. UF and BS. Comparative tests with the addition to the culture medium of plain BS, plain UF and various combinations of UF and BS show that the combinations give an essentially stronger effect on the growth of the adherent cell than what would be expected by adding the effects of the individual components UF and BS. The invention thus provides a composition useful as additive to culture media for the cultivation of adherent cells, said composition comprising - a colostrum fraction prepared by subjecting colostrum, from which fat and cellular debris have been removed by conventional methods, and from which colostrum optionally also casein has been removed by precipitation, to ultrafiltration by using a membrane having a cut off of 100,000 Da and recovering the filtrate, and - a serum component in an amount sufficient to enhance the proliferation of cells after the addition of a suitable amount of said composititon to a culture medium. The invention further provides a culture medium useful for the cultivation of adherent cells, said culture medium comprising - a basal growth medium, - a colostrum fraction prepared by subjecting colostrum, from which fat and cellular debris have been removed by conventional methods, and from which colostrum optionally also casein has been removed by precipitation, to ultrafiltration by using a membrane having a cut off of 100,000 Da and recovering the filtrate, - a serum component, and optionally - additional transferrin and other supplemental agents. The invention further provides a method of promoting proliferation of adherent cells in a culture medium said method comprising contacting an amount of cells with a culture medium according to the invention for a certain period of time. For the cultivation of certain cells, e.g. Vero cells, the transferrin present in the serum may be enough. For other cells such as CHO-Kl-cells, however, the culture medium must be supplemented by additional transferrin. The colostrum fraction is for practical reasons preferably obtained from bovine colostrum. Colostrum from other sources is, however, also believed to be useful for the preparation of the colostrum fraction to be used in this inventio . The serum component is preferably bovine serum, but other sera such as horse sera are also believed to be effective. According to a preferred embodiment of the invention, the colostrum fraction has an endotoxin content of less than 5 EU/ l (units per ml), a total protein content of less than 4.5 mg/ml and an immunoglobulin content of less than 1.2 mg/ml. Particularly preferable are compositions of the invention wherein the colostrum fraction has an endotoxin content of 1.0 EU/ml or less, a total protein content of 2.0 mg/ml or less and an immunoglobulin content of 0.25 mg/ml or less. Such a fraction can be obtained by e.g. by subjecting the defatted colostrum to casein precipitation before the ultrafiltration. The composition of the invention comprises preferably serum component in an amount of at least 0.5 % of said composition. The composition according to the invention can further comprise one or more supplemental agents selected from the group comprising sodium selenite, ethanolamine, β- mercaptoethanol, bovine serum albumin and transferrin. Although the culture medium of this invention can be prepared by adding separately appropriate amounts of colostrum fraction and serum to the basal growth medium, the culture medium is preferably made by adding to the basal growth medium an appropriate amount of a composition redily including the colostrum fraction and serum component. According to a particularly preferred embodiment, the composition further includes added amounts of transferrin and optionally also other supplemental agents. According to a further preferred embodiment, the culture medium has a concentration of the colostrum fraction ranging from about 1 % to about 20 % and a serum concentration that is at least 0.1 % of said culture medium. Most preferably, the culture medium has a concentration of the colostrum fraction ranging from about 5 to 15 % of said culture medium. Although the method of promoting proliferation of cells in a culture medium according to the invention is useful in the cultivation of adherent cells generally it is especially advantageous for the cultivation of adherent cells such as CH0-K1 (Chinese hamster ovary cells) and Vero (African green monkey kidney cells) as these two represent the technically most important adherent cells. EXPERIMENTS The aim of the experiments presented below is to show that a composition of bovine serum (BS) and a bovine colostrum fraction, colostrum ultrafiltrate (UF), can support proliferation of adherent cells. Materials and methods Cells Chinese hamster ovary cells (CHO-K1, ATCC CCL 61) and African green monkey kidney cells (Vero, ATCC CCL 81) were obtained from Flow (Flow Laboratories, Rickmansworth, England) . Cell cultivation The basal media used were Dulbecco's Modified Eagles Essential Medium (DMEM) (for Vero cells) (GIBCO Life Technologies Inc., Paisley, Scotland) and DMEM-F12 (1:1) (GIBCO) (for CHO-K1 cells) supplemented with glutamine (4 mM), penicillin (100 units/ml) and streptomycin (100 μg/ml). Stock cultures were maintained in 75-cm 2 plastic flasks (Cos ar, Cambridge, Mass, USA) supplemented with 10 % FBS (HyClone, Logan, Utah, USA). For subculture, cells growing at mid or late exponential phase were washed with the basal medium and detached from culture flasks by incubation with trypsin (0.05 %)-EDTA (0.53 mM) (TE) (GIBCO). The cells were suspended in the basal and aliquots of the cell suspension were added to media supplemented with known amounts of adult bovine serum (BS) (SIGMA, St. Louis, MO, USA), fetal bovine serum (FBS) or colostrum ultrafiltrate (UF) (for the preparation of UF, see R Pakkanen et al. , Appl Microbiol Biotechnol (1992) 37: 451 - 456) and human (holo)transferrin (5 mg/1) (SIGMA). The cells were plated in the test media into 24-well plates (Costar) at a concentration of 20 000 viable cells/well (1.5 ml/well) and incubated without medium change for different time periods. For cell counting, the culture supernatants were removed and 150 μl 2xTE was added to each well. The plates were incubated at 37°C for about 10 min. Then, the culture supernatants were added back to the corresponding wells and the cells were suspended by pipetting the cultures. Cell counts were done in a haemocytometer using trypan blue exclusion to determine viability. Each counting point represents the average cell concentration of duplicate wells, and each well was counted only once. RESULTS Cell growth in 10 % FBS and in 13 % UF To study the cell growth in UF supplemented DMEM CHO-K1 and Vero cell were first cultured in 10 % FBS or in 13 % UF supplemented with transferrin 5 mg/1 for 14 days. The results (Fig. 1) showed that both the Vero and CHO-K1 cells could not grow in UF supplemented medium. The reason for this was the inability of the cells to attach to plastic in UF. This is in correlation with a previous finding that bovine colostrum contains only a negligible amount of fibronectin, a major attachment factor present in serum (Steimer and Klagsbrun, J. Cell. Biol. (1981) 88: 294-300. Cell growth in 13 % UF supplemented with 0.1-2 % BS To study the effect of a small amount of adult bovine serum on cell growth CH0-K1 and Vero cells were cultured in 13 % UF supplemented with 0.1-2 % BS and transferrin 5 mg/1. The results (Fig. 2) indicated that even 0.33 % additional BS enhance dramatically the growth of both cell lines. 0.1 % additional BS was not effective. In the cultures supplemented with > 0.6 % BS and 13 % the cells were well spread and their morphology resembles to that in 10 % FBS. This suggests that the growth factors provided by UF and the attachment factors provided by BS can support the growth of the adherent Vero and CH0-K1 cells. In 0.33 % FBS CH0-K1 cells tended to form large clusters and only a small cell population was spread. Maximum growth of Vero cells was obtained in 13 % UF supplemented with 1-2 % BS, whereas no significant differences could be observed in CHO-K1 cultures supplemented with 0.6-2 % BS. Cell growth in 1 % BS supplemented with 3.3-13 % UF To optimize further the concentration of UF and BS Vero and CH0-K1 cells were cultured in 1 % BS supplemented with 3.3- 13 % UF and transferrin 5 mg/1. The results (Fig. 3) indicated that the concentration of UF in the mixture was not so critical. No significant differences were detected in the growth of Vero cells in the different mixtures. CHO- Kl cells achieved maximum cell density in 6.7 % UF. Growth promoting activity of BS compared with that of OF supplemented with BS To study the synergistic effect of UF and BS Vero and CHO- Kl cells were cultured in 0.5-2 % BS and in four combinations of BS and UF supplemented with human transferrin. The results (Fig. 4) showed that CHO-K1 cells did not grow in BS and also Vero cells exhibited significantly better growth in different mixtured of UF and BS compared with that in BS supplemented media. Growth promoting activity of FBS compared with that of UF supplemented with BS To compare the growth promoting activity of different mixtures of BS and UF with that of FBS the cells were cultivated in four combinations of UF and BS (supplemented with transferrin) and in 0.5-10 % FBS. The results (Fig. 5) showed that the maximum cell densities obtained in different combinations of UF and BS were about 70-110 % of that obtained in 1-10 % FBS. In 0.5 % FBS cell growth was significantly reduced. Figure 6 shows the number of viable CHO-Kl-cells versus time in a culture medium supplemented with 10 % FBS, 10 % UF and 10 % UF + 65 μg/ml of fibronectin. As can be seen from the Figure, fibronectin added to the UF did not have any effect on the cell growth. The colostrum fraction from which casein not has been removed before ultrafiltration, which fraction also is useful in the composition and culture medium of the invention, is prepared according to the method described in the paper by R Pakkanen et al. referred to above with the only difference that casein is not precipitated before ultrafiltration.

Claims: CLAIMS 1. A composition useful as additive to culture media for the cultivation of adherent cells, said composition comprising - a colostrum fraction prepared by subjecting colostrum, from which part of the fat and cellular debris have been removed by conventional methods, to ultrafiltration by using a membrane having a cut off of 100,000 Da and recovering the filtrate, and - a serum component in an amount sufficient to enhance the proliferation of cells after the addition of a suitable amount of said composititon to a culture medium. 2. The composition according to claim 1 where casein has been removed from the colostrum before the ultrafiltration. 3. The composition according to claim 1 wherein the colostrum fraction is a bovine colostrum fraction and the serum component is bovine serum. 4. The composition according to claim 1 wherein the colostrum fraction has an endotoxin content of less than 5 EU/ml, a total protein content of less than 4.5 mg/ml and an immunoglobulin content of less than 1.2 mg/ml. 5. The composition according to claim 2 wherein the colostrum fraction has an endotoxin content of 1.0 EU/ml or less, a total protein content of 2.0 mg/ml or less and an immunoglobulin content of 0.25 mg/ml or less. 6. The composition according to claim 5 wherein the serum component is present in an amount of at least 0.5 % of said composition. 7. The composition according to claim 6 wherein said composition further comprises one or more supplemental agents selected from the group comprising sodium selenite, ethanolamine, β-mercaptoethanol, bovine serum albumin and transferrin. 8. A culture medium useful for the cultivation of adherent cells, said culture medium comprising - a basal growth medium, - a colostrum fraction prepared by subjecting colostrum, from which fat and cellular debris have been removed by conventional methods, to ultrafiltration by using a membrane having a cut off of 100,000 Da and recovering the filtrate, and - a serum component. 9. The culture medium according to claim 8 wherein casein has been removed from the colostrum before the ultrafiltration. 10. The culture medium according to claim 9 which further comprises additional transferrin and other supplemental agents. 11. The culture medium according to claim 9 wherein the colostrum fraction is a bovine colostrum fraction and the serum component is bovine serum. 12. The culture medium according to claim 8 wherein the colostrum fraction has an endotoxin content of less than 5 EU/ml, a total protein content of less than 4.5 mg/ml and an immunoglobulin content of less than 1.2 mg/ml. 13. The culture medium according to claim 9 wherein the colostrum fraction has an endotoxin content of 1.0 EU/ml or less, a total protein content of 2.0 mg/ml or less and an immunoglobulin content of 0.25 mg/ml or less. 14. The culture medium according to claim 13 wherein the concentration of the colostrum fraction ranges from about 1 % to about 20 % and the serum concentration is at least 0.1 % of said culture medium. 15. The culture medium according to claim 14 wherein the concentration of the colostrum fraction ranges from about 5 to 15 % of said culture medium. 16. A culture medium useful for the cultivation of adherent cells, said culture medium comprising a basal growth medium and a composition according to any one of claims 1 to 7. 17. The culture medium according to claim 16 wherein said composition is present in an amount sufficient to give a serum concentration of at least 0.1 % of said culture medium. 18. The culture medium according to claim 17 wherein said composition is present in an amount sufficient to give a colostrum fraction concentration ranging from about 1 % to about 20 % of said culture medium. 19. The culture medium according to claim 18 wherein said composition is present in an amount sufficient to give a colostrum fraction concentration ranging from 5 to 15 % of said culture medium. 20. A method of promoting proliferation of adherent cells in a culture medium said method comprising contacting an amount of cells with a culture medium according to claim 8 for a certain period of time. 21. A method of promoting proliferation of adherent cells in a culture medium said method comprising contacting an amount of cells with a culture medium according to claim 16 for a certain period of time. 22. The method according to claim 21 wherein the cells are Vero cells. 23. The method according to claim 21 wherein the cells are CH0-K1 cells.

Inventors: AALTO JOUNI UOLEVI, JALONEN HARRY GOESTA, KANTTINEN ARI PERTTU TAPANI, PAKKANEN RAIMO ANTERO, AALTO, JOUNI, UOLEVI, JALONEN, HARRY, GOESTA, KANTTINEN, ARI, PERTTU, TAPANI, PAKKANEN, RAIMO, ANTERO
Applicants: VALIO BIOTUOTTEET OY
Application year: 1994
Main classification: C12N5
IPC codes: C12N5